Process of removing biological contaminants with enzymes from krill

ABSTRACT

Enzymes isolated from krill of the order Euphausiaceae are used to remove biological contaminants. Preferably, a mixture of enzymes including exo-and endopeptidase is isolated. The enzymes can be used in laundering or to clean or debride living tissue. Isolation may be carried out by homogenizing krill and extracting the enzymes with an aqueous medium. The enzymes may be further purified by gel chromatography. After lipids have been removed, the enzymes can be lyophilized for long time storage.

This application is a division of application Ser. No. 082,134 filedAug. 6, 1987, now U.S. Pat. No. 4,801,451, which in turn is acontinuation of application Ser. No. 621,911 filed June 18, 1984, nowabandoned.

Field of the Invention

This invention relates to a composition which contains a digestiveand/or tissue enzyme preparation from an aquatic animal selected fromthe group consisting of animals belonging to the order Euphausiaceae orto the fishes. The composition is used in order to clean living or deadmaterial by degrading and removing therefrom contaminants of biologicalorigin or degradation products of such contaminants. The invention alsoprovides a method for removing said contaminants from said material anda method for the preparation of a composition to be used according tothe invention.

As used in the specification and in the claims, the term enzyme means anactive enzyme if not otherwise specified.

The invention relates to both the therapeutical cleaning of mammals andto the non-therapeutical cleaning in general. The concept cleaning ishence used in its broadest meaning, i.e. cleaning of dead as well as ofliving material such as different types of textiles, hair, furs, skins,plastics, leather, nails, ears, mirrors, glass, porcelain, dentureprosthesis, metals, stones, teeth, facades, downs, works of art such aspaintings, etc. Cleaning also includes cleaning of humans and animals byremoving substances such as pus (purulent exudate), fibrin, coagulatedblood, blood crusts and necrotic tissue. This latter type of cleaning isespecially important for the treatment of wounds, burns and dermatoses,e.g. for the so called enzymatic debridement, but can also be carriedout in other areas of the living body, where the contaminants can occur.The uretra and the urinary bladder are examples of such other areas.

The normal digestive effect of the enzymes in the animals, as well astheir autolytic action in the animals post mortem, are not included inthe term cleaning.

TECHNICAL BACKGROUND

In krill (which belongs to the order Euphausiaceae), a mixture ofdifferent enzymes exists, such as e.g. proteinases (with acidic andneutral-to-alkaline pH-optimun), peptidases (exo- and endopeptidases),lipases, phospholipases, amylases and other carbohydrate degradingenzymes, phosphatases, nucleases, nucleotidases and esterases (T. E.Ellingsen; Biokjemiske Studier over Antarktisk Krill; Dr. ingavhandling; Institute for Teknisk Biokjemi, Norges Tekniske Hogskole,Trondheim (1982). The proteolytic (trypsin-like) activity existing in awater extract from krill has been studied and described (C. S. Chen etal; J. Food Biochem. 2 (1978) p. 349-66)). Different protease activitiesin water extracts from capelin have also been described previously (AGildberg; Autolysis of fish tissue--General aspects; Thesis; Instituteof Fisheries, University of Troms Norway (1982)).

As early as 1913 proposals were made to use enzymes in detergents.Enzyme compositions for cleaning of dead materials, e.g. as laundryagents, have previously been based on different microbial proteases fromthe genus Bacillus. One such protease commonly used is subtilisinderived from Bacillus subtilis strains and marketed among others underthe name Alcalase® (Novo Industri, Copenhagen, Denmark). Differentlipases have also been used for cleaning, especially for enablingdegradation of lipids. In addition to enzymes, such compositions havecontained also different anionic, cationic and neutral detergentstogether with blanchophores, such as perborates. The enzymes usedhitherto have, like enzymes in general, been relatively unstable.

The most important enzyme compositions on the market for debridement ofthe components mentioned above are Strepto-kinase-streptodornase(Varidase®, Lederle Lab., American Cyanamid Company, Wayne, N.J., USA),stabilized crystalline trypsin (Trypur®, Novo Industri, Copenhagen,Denmark) and bovine fibrinolysin combined with deoxyribonuclease(Elase®, Parke Davis & Company, Detroit, Mich., USA). Streptokinase actson necrotic material mainly by its effect on DNA and streptodornase hasa specific fibrinolytic effect. Trypsin acts proteolytically and isextracted from bovine pancreas. Fibrinolysin-deoxyribonuclease is acombination of two enzymes--one fibrin degrading enzyme and one actingon deoxyribonucleic acid which is an important component in pus.

One specific pepsin-like enzyme (pepsin I) from Mallotus villosus(capelin) has been proposed to be used in the medical treatment of burns(Gildberg A; cited above p. 89-90). The use of one specific enzymeacting on one type of substrates is likely to achieve only a limitedbreakdown of the contaminants present in wounds. However, no combinationof this pepsinlike enzyme with other enzymes has been proposed for thetherapeutical cleaning of humans.

The enzyme preparations mentioned above suffer from several drawbacks.Thus, all of them are relatively unstable leading to a rapid decrease intheir activity, either during storage or use. Their activities are oftenlimited to a certain pH-range, e.g. neutral to moderately alkaline pH.Their activities are also in many cases restricted to certaintemperature intervals. At a temperature above +50° C., a rapid loss ofactivity is observed and at room temperatures or normal outdoortemperatures, their activities are low.

The effect of Varidase®, Trypure® and Elase® are relatively poor for itspurpose Only a moderate debriding effect is usually achieved after atreatment over a period of three weeks.

THE OBJECTS OF THE INVENTION

One object of the present invention is to provide an improved cleaningcomposition with respect to the drawbacks mentioned above. Anotherobject is to provide a method for the preparation of an improvedcomposition to be used for the removal of contaminants containingsubstances of biological origin or degradation products from suchsubstances, especially contaminants containing both proteins and lipidsA third object is to provide a new and improved method for cleaningliving or dead material from the contaminants mentioned above,especially for the debridement of fibrin, coagulated blood anddevitalized tissue by degrading these constituents and therebyfacilitating their removal without apparently increasing their watercontent. The invention hence is based on compositions having moreeffective enzymes than the prior art compositions. This is valid for thetotal enzymatic effect on the contaminant in question.

THE INVENTION

These objects are achieved by using a composition containing aneffective amount of an enzyme preparation which is capable of dissolvingcontaminants of biological origin and which derives from the animalsmentioned in the introductory part. Enzyme mixtures from these animalscan be obtained in a high yield and in a simple way. At present the mostpreferred and useful sources for the enzyme preparation are animals ofthe order Euphausiaceae, e.g. antarctic krill (Euphausia superba),Euphausia crystallorophias, and related species and other species ofkrill including Meganyctiphanes norvegica, Tysanoessa inermis and otherrelated species. Among fishes, those of the genus Mallotus, particularlyof the species Mallotus villosus (capelin), are preferred according tothe invention. Among other sources, mackerel may be mentioned.

The most important enzyme activities for the invention appear tooriginate from the digestive tract of the animals. The complex mixtureof different enzyme activities obtained from the animals is probably theexplanation why the composition effects a surprisingly rapid degradationof biological contaminants irrespective of their origin. The enzymes areactive in alkaline, neutral and acid medium, and can be utilized incleaning compositions together with different surfactants (tensides andemulgators), and/or other components such as carriers and additives.

The fact that the composition may contain a mixture of enzymes makes ituseful for the removal of contaminants containing mixtures of substancesselected from the group consisting of lipids, phospholipids, biopolymerssuch as proteins, peptides, nucleic acids, mucopolysaccharides andpolysaccharides, and degradation products of such compounds. Thesecompounds are present in pus, blood crust and necrotic tissues.

According to the invention, particularly good results are obtained ifthe enzymes used have molecular weights within the range of 15 000 to 80000 Daltons or active aggregates of such enzymes. Especially, enzymeshaving molecular weights from about 20 000 to about 40 000 Daltons arepreferred. These ranges apply to enzymes obtained by a water extractionof the homogenized animals, i.e. especially to enzymes being watersoluble during the extraction.

One embodiment of the invention relates to a new enzyme cleaningcomposition and to a method for the preparation thereof. The method ischaracterized in that an enzyme preparation, originating from an animalof the order Euphausiaceae or from a fish, is mixed with, dissolved in,bound to or otherwise combined with one or more water-insoluble orwater-soluble aqueous or non-aqueous carriers, if necessary togetherwith suitable additives. The new composition may be in the form of anointment, a powder, a paste, a cream, a spray, a gel, a liniment, abandage, an oil, a tablet, a syrup, a granulate, a capsule, a tabletetc.

Certain non-sterile homogenous water solutions only consisting of buffersubstances, water as the single solvent, and an enzyme preparation fromthe animals in question are excluded from the concept of the newcomposition. Such water solutions have been described in thepublications cited above. The same applies to lyophilized water extractcontaining no additives.

In the presently most preferred embodiment, the enzymes in thepreparation used are water-soluble and/or have molecular weights in theranges mentioned above.

The invention also relates to a method for removing biologicalcontaminants from living or dead material, e.g. by an enzymaticdebridement. The method is characterized in that such contaminants beingpresent on said material are contacted with an enzyme compositioncontaining an effective amount of enzymes, e.g. a proteolytically and/orlipolytically effective amount of enzymes, originating from the animalsmentioned above, whereafter the composition is removed from saidmaterial together with degraded or dissolved contaminants-. In thepresently most preferred embodiment, the method utilizes enzymes(including their active aggregates) having molecular weights from about15 000 to about 80 000 Daltons or active aggregates of such enzymes.Especially, enzymes of molecular weights from about 20 000 to about 40000 Daltons are preferred. Very great advantages are obtained for theremoval of devitalized substances such as pus, blood crusts and necrotictissue, and fibrin or coagulated blood This is especially valid whenthese substances are removed from a living tissue.

The time period required to degrade and/or dissolve the contaminantsvaries from case to case, but should be selected sufficiently long toallow the enzymes to degrade and/or dissolve the contaminants. Thetreatment can be repeated, if necessary.

The enzyme preparation used may contain a wide variety of differentenzymes e.g. of the types mentioned above. According to the invention itis possible to use compositions in which one or more of these enzymeshave been removed by methods known per se, e.g. by adding an inhibitorspecific for one enzyme or by removing the enzyme in question.

The proteolytic activity at optimum temperature is high in the pH-range5-10. The pH-optimum is in the range 7-9. The temperature range for goodproteolytic activity is 5-70° C. and the optimal temperature is withinthe range of 30-55° C. Activity is maintained down to 0° C.

The temperature and pH-ranges given above may all be utilized fordifferent applications of the invention, although certain potentialembodiments may also utilize other ranges.

The preparation of the enzymes from the animals is performed by wellknown methods. Hence, fresh or freshly frozen animals may by homogenizedand extracted with aqueous medium (e.g. water). The extract obtained maybe lyophilized and stored. The extract can be further purified e.g. byextraction with a lipid dissolving solvent in order to extract lipids.If further purification is required, gel-, ultra- or membrane filtrationmay be carried out. Other purification steps, which are potentiallyuseful, are ion exchange and affinity chromatography. The extraction andthe homogenization should be carried out in the cold below or close to+5° C.

The enzyme preparations which are obtained by a water extraction, may beused directly or, if necessary, after further purification. In mostcases, it is of advantage to lyophilize a preparation from which lipidshave been extracted. In such cases, the powder obtained may be storedfor a long time. Each field of use requires specific compositions orforms thereof. Such forms are known per se. Thus the invention relatesto enzyme compositions of different physical forms, such as lyophilizedenzymes, homogeneous aqueous solutions or the enzyme compositionsmentioned above and claimed to be new. The exact amount of enzymes ineach composition varies from case to case--from the pure lyophilizedextract to very complex detergent compositions. The amount of enzymeshould be effective in degrading and/or dissolving the contaminantsintended, without giving unnecessary adverse effects on the materialfrom which the contaminants are to be removed. The other components inthe new cleaning composition should be selected so that they will notadversely affect it under the condition required for its use.

The amount of the enzyme preparation existing in the final compositionmay vary from 0.0001% (w/w) up to 100%. With respect to proteaseactivity, the final composition may contain from 0.0001 to 0.1 enzymeunits per mg. Depending on the purity of the enzyme preparation used,other ranges may apply. The enzyme units above are given as μmoltyrosine equivalents per min with casein as substrate.

In compositions according to the invention different additives andcarriers may be incorporated. Suitable carriers may be sterile, aqueousand physiologically acceptable salt solutions, organic and inorganicgel-forming materials, e.g. polymeric ones, silicone oils and othersubstances and mixtures thereof providing the desired physicalproperties of the composition. Among additives antimicrobials, perfumes,different anionic, cationic, zwitterionic and neutral surfactants(tensides and emulgators) may be mentioned. Such components are alreadyknown per se in connection with other enzyme compositions for laundringor enzymatic debridement, see e.g. German patent application 2 130 833,GB patent specifications Nos. 1 280 497, 1 296 901 and 1 573 964 andU.S. Pat. Nos. 3,627,688, 3,855,142, 4,212,761, 4,243,546, 4,242,219,4,287,082, 4,305,837 and 4,307,081, which all are incorporated herein byreference.

For treatment of injured living tissues, it is advantageous to use acomposition containing a physiologically and pharmaceutically acceptablecarrier or no carrier at all. Such a composition may be sterile.Sterilization may be carried out e.g. by sterile filtration when thecomposition is a solution. Sterile compositions may also be obtained bymixing sterile components under aseptic conditions.

The different embodiments of the invention will further appear from theclaims, hereby enclosed to the specification. Below, the invention willbe illustrated by different non-limiting examples.

EXAMPLE 1 A. Preparation of a Krill Extract

Krill, Euphausia superba, caught during the Antarctic summer and frozenimmediately and stored for about 2 years at about -80° C., are placed ina room at about +5° C. When the krill is nearly thawed, 25 g of thekrill is mixed with 50 ml of deionized water having a temperature of 0°C. The mixture is homogenized and then centrifuged in the cold (about 0°C.) for half an hour at 12 500 g. The red supernatant is decanted andsaved. The sediment is resuspended in 50 ml deionized water andcentrifuged as above. The new supernatant is decanted and combined withthe supernatant from the first extraction.

In order to remove lipids from the extract 20 ml of CCl₄ is added to thecombined supernatant and homogenized in the cold (0° C.). The mixture iscentrifuged at 2500 g for 15 min in the cold. The water-phase is removedand extracted once more with CCl₄ and centrifuged as described above.The water-phase is used according to 1B, where it is referred to as thewater extract.

B. Further Purification by Gel Chromatography

20 ml of the water extract from A is chromatographed on Sephadex® G-100(dextran crosslinked with epichlorohydrin, Pharmacia Fine Chemicals AB,Uppsala, Sweden) in a column having a diameter of 3,1 cm and a height of69 cm. The column is equillibrated and eluted (30 ml per hour) withTris-HCl buffer (0.05M, pH 7.5) at +5° C. Fractions are collected. Theelution profile is monitored spectrophotometrically by measuring the UVadsorption at 280 nm and the proteolytic activity of the fractions aredetermined separately. The enzymatically active fractions are pooled anddialyzed against deionized water. Finally, the pooled fractions arelyophilized and used according to the invention.

The proteolytic activity in the fractions and in the lyophilizedpreparation is determined by using hemoglobin and/or casein assubstrates (Rick, W I; Methods of Enzymatic analysis; Ed Bergmeyer, H U;Vol 2, p. 1013-23 (1974); Academic Press, New York). By carrying out gelchromatography, enzyme activity is mainly recovered from fractionscorresponding to the molecular weights of 20 000 to 40 000 Daltons.

EXAMPLE 2 Preparation of a Capelin Extract

Capelin (Mallotus villosus), caught off the coast of Finnmark (Norway)in the month of September, was frozen and stored at -20° C. for oneyear. The frozen capelin was then placed at 5° C. After 24 hours theintestines including the digestive tract were removed from the partiallythawed capelin. 25 g of intestines were mixed with 50 ml of deionizedwater and homogenized at 0° C. The mixture was subsequently centrifugedat 12500 g for half an hour. The partially cloudy supernatant isdecanted and saved. The sediment is resuspended in 50 ml deionized waterand centrifuged as above. The new supernatant is decanted and combinedwith the supernatant from the first extraction.

In order to remove lipids from the extract 20 ml of CCl₄ were added tothe combined supernatant and homogenized in the cold (0° C.). Themixture was centrifuged at 2500 g for 15 min in the cold. Thewater-phase was removed and extracted once more with carbontetrachlorideand centrifuged as described above. The water-phase was finallylyophilized.

EXAMPLE 3 Compositions Containing Enzymes from Mallotus Villosus

A. 10 mg of the lyophilized enzyme preparation from capelin (accordingto example 2) are mixed with 0,4 mg calcium acetate and amylum resorb ad1 g (Biosorb®; Arbrook, Kirkton Campus, Livingston, Scotland).

The homogenous powder so obtained is agitated in 10 g of physiologicalsodium chloride solution (saline) and can be used for wet dressings.

B. 10 mg of the lyophilized capelin preparation (from example 2) aremixed with a polyethylene glycol gel (containing equal parts of Macrogol600 and Macrogol 800 (Hoechst, Federal Republic of Germany)) ad 1 g. A1% enzyme composition having a semi solid, homogenous consistency isobtained.

EXAMPLE 4 Composition Containing Krill Enzymes in a Solution

10 mg of a lyophilized enzyme preparation (from example 1B) are mixedwith 0.4 mg calcium acetate and amylum resorb ad 1 g. The homogenouspowder so obtained is shaken with 10 g saline and the compositionobtained is used in order to soak a sterile compress having the size of3×3 cm and containing 4 layers of a gauze. The wet compress is appliedon a necrotic wound. A gauze is wound two turns around the compress inorder to affix it. This bandage is then further affixed with an adhesiveplaster. The compress is soaked every fourth hour with the composition.In order to limit the application only to that part of the compresswhich covers the wound, the mixture is applied by the use of aninjection cannula having a coarse apex. Once a day, the whole dressingis removed and replaced by a fresh dressing. During this operationdegraded and disintergrated necrotic parts must be removed mechanically.In the specific treatment carried out, the wound appeared clean after 1week.

EXAMPLE 5 Effect of Krill Enzymes on Necrotic Materials

In order to examine the effect of the krill enzymes on necroticcomponents existing on the skin of humans, the following experiment wascarried out:

Necrotic material was applied on the intact skin of the upper side ofthe right hands of two persons. The necrotic material was from a woundin a leg. The material consisted of devitalized collagen tissue,coagulated blood, fibrin, pus and crusts. The amount applied for eachperson was 0.5 g.

The necrotic material on each person was covered with a compress (area10 cm²), which had been soaked with the krill enzyme compositionprepared as in example 4. Each compress was covered with a gauze. After4 hours both of the compresses were nearly completely dry and thereforemore of the enzyme composition had to be added. After 12 hours thecompresses were removed and the necrotic material remaining on each handwas weighed separately. The material showed characteristic signs ofdegradation and had decreased their weights with about 25%. The skin,contacted with the necrotic material during the test, were unaffected.No negative symptoms were experienced by the test persons.

EXAMPLE 6 In Vivo Test on a Person Having a Minor Necrotic Wound,

A person having a minor ulcer containing necrotic material on his rightforefinger, treated himself over a period of 5 days with dailyapplication of a 1% krill enzyme solution (prepared as in example 4).The wounded area tolerated the enzyme composition well and showed asignificant tendency to become clean and to epithelialize. No adverseeffects were observed.

EXAMPLE 7 The Cleaning Effect on Lipid Rich Seborrhoic Skin by KrillEnzymes Combined with an Amphoteric Tenside

A conventional detergent was used consisting of 18,00% (w/w)alkylimidazoline, 1,90% (w/w) lauryl-polypeptid condensate, 1,70% w/w)undecylenyl-polypeptid condensate, 28,00% (w/w) modified alkyl ethersulfate buffered with fat substances (lanoline), 1,40% (w/w)coconut-amine sarcosinate, 1,90% (w/w) undecylene acid, 2,00% (w/w)undecylene-monoethanolamide sulfosuccinate, lactic acid q.s. pH 6,4 andaqua bi dest and 100,00% (w/w).

An amount of the lyophilized krill enzyme preparation (from example 1B)was added to the detergent giving a 1% (w/w) enzyme detergentcomposition. An area of 10 cm² of skin in regio sternal is of twopersons, having an apparent seborrhoea oleosa in this region, wasselected for testing. These areas were washed twice a day during fivedays with the enzyme detergent composition Samples from the same areawere taken both before and after the washing period. The samples werecollected and analysed photometrically according to Schaeffer H andKuhn-Bussius H. (Arch. Klin. u. exper. Dermatol. 238 (1970) p. 429).

The result showed a significant decrease in the transmission value forboth of the two persons examined. Hence, this indicates a degradation ofthe lipid of the skin by the enzyme composition used. Comparatively verylittle effect was obtained, when the two persons used only detergent inthe abscence of enzymes.

EXAMPLE 8 Comparative Washing Experiments on Textile. A ComparisonBetween Enzyme From E.Superba (Krill), Alcalase® And Destilled Water

Equal pieces (5×5 cm) were cut from a homogenous part of a cloth. Thecloth was two folded having one side of silk and the other of a weftconsisting of a mixture of cotton and synthetic fibers. Six of thepieces were stained with blood, six with milk and six with indian ink.To each of six Ehrlenmeyer flasks, 100 ml of an aqueous solution (0.5%w/v) of the lyophilized krill enzyme were added (the lyophilized krillenzyme was obtained from example 1B). To each of six other Ehrlenmeyerflasks, 100 ml of a 0.5% (w/v) of Alcalase® (Novo Industri, Copenhagen,Denmark) solution in aq. dest were added. 100 ml aq. dest were added tosix other Ehrlenmayer flasks.

To each of two flasks containing the krill enzyme solution, to each oftwo flasks containing the Alcalase® solution and to each of two flaskscontaining aq. dest, one of the pieces stained with blood was added.Analogously, the other stained pieces were added to the remainingflasks, so that only one piece was present in each flask. This meant,that each of the compositions were allowed to act in duplicate on clothstained with blood, milk and indian ink. Thereafter, the flasks wereagitated on a shaking water bath for one hour at +45° C. After thistreatment, the washing liquid was decanted and 100 ml aq. dest wereadded to each flasks. The flasks were then sealed with a rubber plug andagitated heavily for one minute. Thereafter the pieces of cloth werecollected and washed slowly in flowing tap water for 10 minutes. Thepieces were folded in clean white towels and allowed to dry.

The effect of the washing, i.e. the brightness of the cloth, was thendetermined by a double blind test, i.e. the examiner did not know whichwashing composition each piece had been treated with. The washingeffect, i.e. the brightness and cleanness of the cloth, was evaluatedvisually in the order of precedence; 0=completely clean cloth;1=insignificant amounts of remaining dirt; 2=moderate amount ofremaining dirt; 3=considerable amounts of remaining dirt. The resultsfrom the washings are presented in Table 1.

                  TABLE 1                                                         ______________________________________                                        The washing effect of 0.5% (w/v) krill enzyme in aq.                          dest; 0.5% (w/v) Alcalase ®  in aq. dest; and aq. dest                    without addition of enzymes.                                                  0.5% krill-      0.5% Alcalase ®                                          enzyme in aq. d. in aq. d.    aq. dest alone                                  Contami-                                    Cloth                             nants   Cloth I Cloth II Cloth I                                                                             Cloth II                                                                             Cloth I                                                                             II                                ______________________________________                                        Blood   1       1        2     2      2     3                                 Milk    1       1        2     1      2     2                                 Indian ink                                                                            2       2        2     2      3     3                                 ______________________________________                                    

From Table 1 it is seen that the enzyme composition from krill is moreeffective than Alcalase® or aq. dest. Similar tests can be performedwith an analogous composition containing capelin enzymes.

EXAMPLE 9 Degradation of Fibrin, Necroses and Coagulated Blood A. Fibrin

Fibrin was dissected into pieces varying in weight from 0.2-0.3 g. Foreach enzyme composition used, 20 test tubes were numbered and to each ofthem one piece of preweighed fibrin was added. To the 20 tubes of eachcomposition, equal amounts of the enzyme composition were added.Varidase® was used in the dilution of 1:20 (w/v based on the commercialpreparation) in destilled water. Trypure® was used in the concentrationof 1:15 (w/v based on the commercial preparation) in saline. Thepreparations of the other enzymes used were diluted in saline and theconcentrations of the composition so obtained are given in Table 2. Thekrill enzymes and capelin enzymes used were the lyophilized preparationsobtained according to example 1B and 2, respectively. The study wasperformed at a temperature of +33° C. which is the same as thetemperature in wounds. After 12 hours and 24 hours, respectively, thereactions were stopped and the fibrin remaining was collected and kepton a wet filter paper for 60 seconds and weighed. Papain, Ficin andPankreatin were all obtained from E. Merck, Darmstadt, Germany.

                  TABLE 2                                                         ______________________________________                                        The influence of different enzyme compositions on fibrin.                     Concentrations are given in % w/v.                                            ______________________________________                                        -           denotes  0-25% decrease in weight                                 --          denotes 26-50% decrease in weight                                 ---         denotes 51-75% decrease in weight                                 ----        denotes 76-100% decrease in weight                                ______________________________________                                                        Reading after                                                 Enzyme            12 hour    24 hour                                          ______________________________________                                        Trypure ®     ----       nothing left                                     Varidase ®    --         --                                               Ficin 1%          -          -                                                Papain 1%         --         --                                               Pankreatin ® 1%                                                                             ---        ---                                              Krill enzymes 1%  ----       nothing left                                     Capelin enzymes 1%                                                                              ----       nothing left                                     ______________________________________                                    

Fibrin was dissolved in the shortest time by the compositions containingkrill enzymes, capelin enzymes and Trypure® and in the order given.Varidase® and Alcalase® were similar to ficin, and papain had theweakest effect.

B. Degradation of Necroses

Necroses were dissected in pieces of 0.2-0.3 g. For each enzymecomposition used, 20 test tubes were numbered and to each of these tubesone piece of the preweighed necroses were added. To the 20 test tubes ofeach composition, equal amounts of each enzyme composition were added.The compositions used are given in Table 3 and their concentrations aregiven as % (w/v) in saline. Varidase® and Trypure® were dilutedaccording to example 8A. After time intervals of 12, 24, 36 and 72hours, the degradation was stopped and the necroses were collected andweighed.

                  TABLE 3                                                         ______________________________________                                        The influence of different enzyme compositions on necroses.                   Concentrations in % are calculated on a w/v basis.                            ______________________________________                                        ----         decrease in weight                                                                            76-100%                                          ---          decrease in weight                                                                            51-75%                                           --           decrease in weight                                                                            26-50%                                           -            decrease in weight                                                                            1-25%                                            0            status quo      0%                                               +            increase in weight                                                                            1-25%                                            ++           increase in weight                                                                            25-50%                                           +++          increase in weight                                                                            51-75%                                           ++++         increase in weight                                                                            75-100%                                          Enzyme composition                                                                          Reading after                                                           Conc.     12 hrs  24 hrs 36 hrs 72 hrs                                ______________________________________                                        Varidase ®                                                                        1:20 (w/v)                                                                              +++     +++                                                 Trypure ®                                                                         1:15 (w/v)                                                                              ++      ++                                                  Ficin   0.5%      0       0      0      -                                             1.0%      +       0      --     ---                                           2.0%      +       -      -      --                                    Papain  0.5%      0       +             +                                             1.0%      0       +             +                                             2.0%      +       +      +      +                                     Alcalase ®                                                                        0.5%      0       -      0      -                                             1.0%      -       -      --     --                                            2.0%      -       -      -      -                                     Krill   0.5%      -       --     --     --                                    enzymes 1.0%      --      ---    ---    ---                                           2.0%      -       ---    ----   ----                                  Capelin 0.5%      --      --     ---    --                                    enzymes 2.0%      -       --     --     ---                                   Pan-    0.5%      0       ++     ++     ++                                    kreatin ®                                                                         1.0%      +       +++    +++    +++                                           2.0%      ++      ++++   +++    +++                                   ______________________________________                                    

Apparently, Alcalase®, krill enzymes and capelin enzymes are really ableto dissolve necroses and do not increase the water content thereof likeVaridase®, Trypure®, and saline. However, when regarding their influenceon the necroses, papain and Pankreatin® show the same tendencies as thecompositions on the market, i.e. an increase in weight due to anincrease in water content. Ficin has an effect similar to but weakerthan Alcalase®, the krill enzymes and the capeline enzymes.

C. Degradation of Coagulated Blood

Coagulated blood was dissected and portioned into test tubes arranged asin example 9A and B. The enzyme preparations used were dissolved insaline, and the concentrations (% w/v) of the compositions so obtainedare given in Table 4. After the time intervals 12 and 24 hours,respectively, the degradation was stopped, and the blood coagels werecollected and weighed.

                  TABLE 4                                                         ______________________________________                                        The influence of different enzyme compositions on coagulated                  blood.                                                                        ______________________________________                                        -           decrease in weight                                                                             0-25%                                            --          decrease in weight                                                                             26-50%                                           ---         decrease in weight                                                                             51-75%                                           ----        decrease in weight                                                                             76-100%                                          ______________________________________                                                           Reading after                                              Enzyme composition                                                                          conc.      12 hrs    48 hrs                                     ______________________________________                                        Varidase ®                                                                              1:20 (w/v) -         --                                         Saline        0.9%       -         -                                          Ficin         1.0%       -         --                                         Papain        1.0%       -         -                                          Alcalase ®                                                                              1%         -         -                                          Pankreatin ®                                                                            1.0%       --        --                                         Krill enzymes 1.0%       ---       ----                                       Capelin enzymes                                                                             1.0%       ---       ---                                        ______________________________________                                    

From Table 4 it is seen that for coagulated blood, the krill enzymeswere superior to the capelin enzymes, which both showed an effectsuperior to the other enzyme compositions investigated.

EXAMPLE 10 Studies of Different Enzyme Compositions on Healthy Skin

Different solutions of ficin, papain. Alcalase®, Pankreatin®, krillenzymes and capelin enzymes--all at concentrations of 1% (w/v)--wereapplied (0.3 ml) on the healthy skin of two experimental persons andcovered by a thin bandage. No sign of inflammation could be observed onthe skin after 12 hours. The same observation was found when usingVaridase® and Trypure®. Hence, the enzymes apparently only attacknecrotic tissues in the concentration ranges used in the tests.

We claim:
 1. In a cleaning process for removing biological contaminantsby the application of enzymes to the contaminants; wherein theimprovement comprises the application of a biological contaminantremoving effective amount of enzymes comprising a mixture of exo- andend-peptidases isolated from krill of the order Euphausiaceae.
 2. Aprocess according to claim 1, in which the enzymes isolated from krillof the order Euphausiaceae have molecular weights in the range of 15,000to 80,000 dalton.
 3. A process according to claim 1, in which theenzymes isolated from krill of the order Euphausiaceae additionallycomprise proteinases.
 4. A process according to claim 3, in which theenzymes are isolated by a process which comprises homogenizing the krilland extracting the enzymes from the homogenized krill with an aqueousmedia.
 5. In a laundering process for removing biological contaminantsby the application of enzymes to the material being laundered; whereinthe improvement comprises the application of a biological contaminantremoving effective amount of enzymes comprising a mixture of exo- andendo-peptidases isolated from krill of the order Euphausiaceae.
 6. Aprocess according to claim 5, in which the enzymes isolated from krillof the order Euphausiaceae have molecular weights range of 15,000 to80,000 dalton.
 7. A process according to claim 6, in which the enzymesisolated from krill of the order Euphausiaceae additionally comprisesproteinases.
 8. A process according to claim 7, in which the enzymes areisolated by a process which comprises homogenizing the krill andextracting the enzymes from the homogenized krill with an aqueous media.